Method of preparing mesenchymal-like stem cells and mesenchymal-like stem cells prepared thereby

Abstract

Disclosed is a method for effectively preparing mesenchymal-like stem cells, the method including: preparing human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines; inducing differentiation of the human pluripotent stem cells to produce embryoid bodies and selecting cystic embryoid bodies therefrom; loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells therefrom; isolating only monolayer-shaped cell clusters from the mesenchymal-like stem cells passing through the cell-permeable 3D culture unit; and uniformizing sizes of the monolayer-shaped cell clusters, in which the mesenchymal-like stem cells have anti-inflammatory efficacy and immunosuppression. Further, disclosed are mesenchymal-like stem cells prepared by the preparation method, a transporter or a therapeutic composition including the mesenchymal-like stem cells, and a composition for prevention or treatment of diseases that includes an active ingredient or exosomes secreted from the mesenchymal-like stem cells.

What is claimed is: 
     
         1 . A method of preparing mesenchymal-like stem cells, the method comprising:
 (a) preparing human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines;   (b) inducing differentiation of the human pluripotent stem cells to produce embryoid bodies and selecting cystic embryoid bodies therefrom;   (c) loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells therefrom;   (d) isolating only monolayer-shaped cell clusters from the mesenchymal-like stem cells passing through the cell-permeable 3D culture unit; and   (e) uniformizing each of longitudinal and transverse sizes of the monolayer-shaped cell clusters to 100 μm to 500 μm, and culturing the mesenchymal-like stem cells,   wherein the mesenchymal-like stem cells have anti-inflammatory efficacy and immunosuppression, and express CD90 and SOX2 at a level of 95% or greater.   
     
     
         2 . The method of  claim 1 , wherein the cell-permeable 3D culture unit is made of at least one selected from a group consisting of nylon, fiber, polyethylene, polypropylene, graphene, titanium, copper, nickel, silver, gold and platinum. 
     
     
         3 . Mesenchymal-like stem cells derived from human pluripotent stem cells prepared by the method of  claim 1 , wherein the mesenchymal-like stem cells express MMP-1 protein and HGF protein at a higher level than bone marrow-derived mesenchymal stem cells express MMP-1 protein and HGF protein, express CD95 at a lower level than the bone marrow-derived mesenchymal stem cells express CD95, and express CD90 and SOX2 at a level of 95% or greater. 
     
     
         4 . The mesenchymal-like stem cells of  claim 3 , wherein the mesenchymal-like stem cells express the MMP-1 protein at a level higher by at least 15 times than bone marrow-derived mesenchymal stem cells express the MMP-1 protein, wherein the mesenchymal-like stem cells express the HGF protein at a level higher by at least 2 times than bone marrow-derived mesenchymal stem cells express the HGF protein. 
     
     
         5 . The mesenchymal-like stem cells of  claim 3 , wherein the mesenchymal-like stem cells express CD95 at a level lower by at least 15 times than bone marrow-derived mesenchymal stem cells express CD95. 
     
     
         6 . The mesenchymal-like stem cells of  claim 3 , wherein the mesenchymal-like stem cells express an inflammation-regulating gene at a level higher by 2 times to 100 times or greater than bone marrow-derived mesenchymal stem cells express the inflammation-regulating gene, and
 the inflammation-regulating gene comprises at least one selected from a group consisting of Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cx3c11, Ptger4, Acp5, Bcr, Socs5, and Mdk.   
     
     
         7 . The mesenchymal-like stem cells of  claim 3 , wherein the mesenchymal-like stem cells co-express Gata3, Adora2a, and Gps2 genes. 
     
     
         8 . The mesenchymal-like stem cells of  claim 3 , wherein the mesenchymal-like stem cells express an immunosuppression gene at a level higher by 2 times to 100 times or greater than bone marrow-derived mesenchymal stem cells express the immunosuppression gene, and
 the immunosuppression gene comprises at least one selected from a group consisting of Gata3, Gps2, Psma1, Apoe, Foxf1, Tek, Cx3c11, Ptger4, Bcr, Socs5, and Mdk.   
     
     
         9 . The mesenchymal-like stem cells of  claim 3 , wherein the mesenchymal-like stem cells co-express Gata3, Gps2, and Psma1 genes. 
     
     
         10 . A therapeutic composition comprising the mesenchymal-like stem cells of  claim 3 , wherein the therapeutic composition comprises one selected from a group consisting of a cellular therapeutic composition, a cellular gene therapeutic composition, a tissue engineering therapeutic composition, an anti-inflammatory therapeutic composition, an immune therapeutic composition, and a composition for preventing or treating cancer. 
     
     
         11 . The therapeutic composition of  claim 10 , wherein the therapeutic composition further comprises an active ingredient or exosomes secreted from the mesenchymal-like stem cells. 
     
     
         12 . The therapeutic composition of  claim 10  or  11 , wherein the therapeutic composition prevents or treats at least one disease selected from a group consisting of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft versus host disease, lower extremity ischemia disease, Burger's disease, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injury caused by various movements, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage from burns, skin damage from ultraviolet light, and inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system disease. 
     
     
         13 . A transporter comprising the mesenchymal-like stem cells of  claim 3 , wherein the transporter carries a pharmaceutical composition therein. 
     
     
         14 . A composition for prevention or treatment of a disease, the composition comprising an active ingredient or exosomes secreted from the mesenchymal-like stem cells of  claim 3 . 
     
     
         15 . The composition of  claim 14 , wherein the disease comprises at least one selected from a group consisting of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft versus host disease, lower extremity ischemia disease, Burger's disease, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injury caused by various movements, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage from burns, skin damage from ultraviolet light, and inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system disease. 
     
     
         16 . A cosmetic composition comprising an active ingredient or exosomes secreted from the mesenchymal-like stem cells of  claim 3 .